Selective expansion of target cell populations

ABSTRACT

The invention provides a method of selective expansion of a predetermined target population of cells, including (a) introducing a starting sample of cells into a growth medium; (b) causing cells of the predetermined target cell population to divide; and (c) contacting the starting cells with a selection element, comprising a plurality of selective binding molecules with specific affinity either for target cells or for non-target cells to select cells of said predetermined target population from other cells in the growth medium. The invention also provides systems for selective expansion and methods of treating patients with cell populations and products.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of U.S. Ser. No.08/446,165, filed May 19, 1995.

BACKGROUND OF THE INVENTION

[0002] This invention relates to systems for expansion of cellpopulations.

[0003] The origin of all the cells in blood and in the immune system isthe hematopoietic stem cell (HSC). Each HSC has the potential todifferentiate into at least eight separate blood cell lineages withinthe myeloid and lymphoid blood cell compartments. It has been estimatedthrough successive generational analysis that one HSC has the potentialto produce up to fifty million differentiated progeny. See U.S. Pat. No.5,061,620, the disclosure of which is incorporated herein by referencein its entirety.

[0004] This enormous potential could be exploited if, starting from asmall number of HSCs, a large pool of HSCs could be grown in culturewithout significant differentiation during expansion. This pool of HSCscould then be used to restore or supplement an immune system and/orblood forming system compromised by, e.g., radiation or chemotherapy.The pool would also be a valuable tool in the design, development andtesting of diagnostic and therapeutic agents used in the treatment ofimmune system and/or blood forming disorders.

[0005] Efforts have been made to develop a system that would grow HSCsex vivo and control cell proliferation and differentiation. Typically,these efforts have involved batch culture of a mixed population of cellsthat have been initially separated from a relatively large volume ofblood.

SUMMARY OF THE INVENTION

[0006] The invention is based on our discovery that a predeterminedtarget population of cells, in particular renewable cells, e.g.,relatively undifferentiated cells including HSCs, can be clonogenicallyexpanded in a system that either (a) positively selects for cells of thetarget population, or (b) negatively selects out non-target cells.Selection of this type occurs concurrently with cell growth orintermittently during cell growth. Advantageously, by selectivelycontrolling the relative populations of cells in the system, theinvention allows greater expansion of the target population. Thisselective population control reduces feedback inhibitions, influencesfactor and substrate consumption rates, and minimizes other limitingfactors that tend to occur in conventional batch cultures.

[0007] In one aspect, the invention features a method of selectiveexpansion of a predetermined target population of cells that includes:(a) introducing a starting sample of cells into a growth medium; (b)causing cells of said predetermined target cell population to divide;and (c) contacting the cells in the growth medium with a selectionelement, comprising a plurality of selective binding molecules withspecific affinity for a predetermined population of cells, so as toselect cells of said predetermined target population from other cells inthe growth medium. The selection element may use positive selection (theselective binding molecules are specific for target cells), or negativeselection (the selective binding molecules are specific for non-targetcells). The method may also include contacting the starting cells with areverse selection element employing the opposite type of selection.

[0008] In preferred embodiments, the starting sample of cells includestarget cells, and the expansion is clonogenic. Alternatively, thestarting sample of cells includes progenitors of said target cells. Insome preferred embodiments, the selection element comprises a solidsupport to which said selective binding molecules are bound. The growthmedium can be disposed in or caused to flow through a chamber. Thegrowth medium may also be caused to recycle through the chamber, flowingfrom an inlet, through the chamber, to an outlet of the chamber, andreturning from the outlet to the inlet via a conduit. It is furtherpreferred that the oxygen saturation of the growth medium be regulatedto be from 0% to 20% relative to the solubility of oxygen in said fluidat equilibrium with air at 37° C. and 1 atm pressure.

[0009] The invention also features a method of selective expansion of apredetermined target population of cells including: (a) introducingfluid containing a plurality of cells into a growth medium; (b) causingcells of said predetermined target cell population to divide; and (c)selecting cells of said predetermined target population from other cellsin the growth medium; wherein steps (b) and (c) are carried outsubstantially simultaneously.

[0010] The invention also features a system for continuous selectiveexpansion of a predetermined target population of cells. The systemincludes (a) a growth medium for supporting cell division; (b) a chamberfor receiving said growth medium; and (c) a selection element,positioned to contact said growth medium during or after cell division.The selection element includes a plurality of binding sites bearing aselective binding molecule. The selective binding molecule can have (i)a specific affinity for cells of said predetermined target cellpopulation or (ii) a specific affinity for non-target cells andsubstantially less affinity for target cells. If desired, the system canfurther include a reverse selection element having the opposite type ofaffinity.

[0011] One system of the invention for continuous selective clonogenicexpansion of relatively undifferentiated cells includes: (a) a tubecontaining a plurality of beads of a size which permits a plurality ofthe undifferentiated cells to grow thereon, the beads bearing on theirsurfaces a plurality of selective binding molecules capable of bindingto a surface antigen present on the relatively undifferentiated cells,wherein the surface antigen is not present on relatively differentiatedcells; (b) means for continuously providing nutrients to the relativelyundifferentiated cells growing on the beads, wherein the nutrients aredelivered via a fluid which flows through the tube and past the beads sothat the relatively undifferentiated cells in the tube divide and atleast a portion of relatively undifferentiated cells exit the tube withthe fluid; and (c) means for continuously harvesting the portion of therelatively undifferentiated cells that exit the tube.

[0012] The invention can be used to provide stem cells (HSCs) useful forenhancing the immune system of a patient. The patient's blood or bonemarrow is withdrawn (or an allogeneic stem-cell containing sample isprovided); stem cells are expanded and harvested according to theinvention; and then those cells are re-introduced into the patient,where they will facilitate enhancement or reconstitution of thepatient's immune and/or blood forming system.

[0013] Preferably, the sample taken from the patient is relativelysmall, e.g., less than about 100 to 200 ml, to minimize trauma to thepatient. The preferred potency and dosage of the undifferentiated cellsto administer to the patient, and duration of administration, will varydepending upon the condition of the patient's immune or blood formingsystem, but would generally be expected to be in the range of from about100 to 1×10⁶ cells/kg body wgt/dose/day.

[0014] Alternatively, the invention can be used to provide to a patienta predetermined population of relatively differentiated cells, byproviding a sample containing a population of cells which cells are theprogenitor to the predetermined population, and using the system of theinvention to cause the progenitor cells to proliferate and differentiateto form the predetermined population of cells, e.g., by providing thecells with a growth factor which will cause differentiation. Forexample, the differentiated cells may be lymphoid precursors, myeloidprecursors or erythroid precursors. The invention can also be used toprovide to a patient a therapeutic compound produced by a population ofcells by using the system of the invention to proliferate cells of thepopulation and to cause the population to produce the substance.

[0015] The term “continuous,” as used herein, refers to a process whichproceeds substantially constantly, with dividing cells being removedfrom the system shortly after they are born, rather than remaining inculture as in a conventional batch process. This term, as used herein,does not imply that the process is necessarily a steady state process,although in some preferred embodiments steady state may potentially bereached.

[0016] The term “specific affinity,” as used herein, refers to atendency to bind a surface molecule or feature that is present on adistinct population of cells and absent on cells not of the population.Examples of such surface molecules or features include but are notlimited to cell adhesion molecules, antigens, carbohydrates andfunctional or non-functional receptors.

[0017] The term “non-specific interaction,” as used herein, refers tointeractions which interfere with and/or reduce the efficiency ofdesired specific interactions.

[0018] The invention provides tremendous potential for continuouslong-term production of cell populations which can be supplied to apatient or other user of the cells almost as soon as the cells are born(or frozen as soon as they are harvested and supplied in frozen form atany desired time). The system can be used as a research tool forstudying the effects of biopharmacological agents, growth factors,mitogens and the like, and also as a diagnostic tool, e.g., to gauge thehematopoietic potential of a patient.

[0019] The invention can be used not only to proliferate relativelyundifferentiated cells, but also to produce populations of other cellssimply by selecting the appropriate growth factor to supply to thesystem during expansion, and to produce desired cell by-products, e.g.,those which could be administered as therapeutic compounds to a patient.Because the initial cell sample can be autologous, the cell populationsor cell by-products produced are likely to be readily accepted by thepatient from whom the cell sample was obtained.

[0020] Because the contents of the system can be frozen, a sample can betaken from a patient, introduced into the system, and then saved for aprolonged period for later use when needed, e.g., when the patient'simmune system or blood forming system is challenged.

[0021] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a highly enlarged diagrammatic view illustrating amethod of positive selection of a target cell.

[0023]FIG. 1a is a highly enlarged diagrammatic view illustrating amethod of negative selection (selection of a non-target cell).

[0024] FIGS. 2-2 d are schematic diagrams illustrating alternative modesof operation of systems according to different embodiments of theinvention.

[0025]FIG. 3 is a somewhat schematic cross-sectional side view of abioreactor suitable for use in the continuous mode of operation shown inFIG. 2c or the recycle mode shown in FIG. 2d.

[0026]FIG. 3a is a highly enlarged, diagrammatic view of a bead used inthe bioreactor of FIG. 3.

[0027]FIG. 4 is a schematic diagram showing the bioreactor in use forcell proliferation in the continuous mode of operation.

[0028]FIG. 5 is a schematic diagram showing the bioreactor duringinitial cycling with reagent.

[0029]FIG. 5a is a schematic diagram showing the bioreactor during arinse cycle subsequent to cycling with reagent.

[0030]FIG. 6 is a schematic diagram showing a system including a recycleloop and both positive and negative selection elements.

[0031]FIG. 7 is a schematic flowchart showing a method, according to oneembodiment of the invention, of treating a patient with HSCsproliferated according to the invention.

[0032]FIG. 8 is a graph illustrating the effect on nonspecificinteraction of treating the bead surface with, alternatively, plasma orfetal bovine serum.

[0033]FIGS. 9 and 9a are graphs illustrating, respectively, the resultsobtained from the experiments described in Examples 1 and 2.

[0034]FIG. 10 is a schematic illustration of a plurality of bioreactorsof the invention arranged in series to allow the cells harvested fromone bioreactor to be expanded, differentiated, or used to produce a cellby-product in another bioreactor downstream therefrom.

[0035]FIGS. 11 and 11a are graphs illustrating the increase in CD34+cells and leukocytes, respectively, in the experiments described inExamples 4 and 5.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0036] As discussed above, the invention broadly features a method ofsubstantially continuously proliferating cells of a desired targetpopulation by providing a system containing a nutrient medium in whichcell proliferation can occur, and selecting cells of the targetpopulation from non-target cells in the system, concurrently withproliferation, intermittently during proliferation or followingproliferation. Cell proliferation and cell selection can be carried outusing an almost infinite variety of different techniques and settings,of which only a few are described below by way of example. Many othertechniques will be readily perceived by those skilled in the art.

[0037] All of the preferred techniques, however, are based on theconcepts of positive selection (providing a selection element having anaffinity for, i.e., “selecting”, target cells) and negative selection(providing a selection element having an affinity for, i.e.,“selecting”, non-target cells). These two techniques, used alone or incombination, allow unwanted cells to be removed from the system andtarget cells to be harvested whenever desired.

[0038] An example of a positive selection technique is illustrateddiagrammatically in FIG. 1. Briefly, one or more biotintylatedantibodies, specific for the target cells, and avidin are sequentiallyintroduced into the system. After a specified incubation time anybiotintylated antibody and avidin which have not formed a complex withthe target cells are rinsed away. Biotintylated dextran-iron is thenadded to the cell suspension. The biotintylated dextran-iron reacts withthe Avidin/Biotintylated Antibody/Antigen Complex. This suspension isthen passed through a magnetic field. Positively selected cells remainin the magnetic field while cells which do not have the iron conjugatedcomplex are removed. After capture and rinsing the magnetic field isremoved and the positively selected predetermined target population isreturned to the nutrient medium.

[0039] An example of a negative selection technique is illustrateddiagrammatically in FIG. 1a. Briefly, one or more anti-dextranconjugated antibodies specific for a predetermined population which isnot of the predetermined target population is introduced into theculture. After a specified incubation time the cell suspension is passedthrough a column containing dextran coated glass beads. AnAntigen/Antibody/Anti-dextran/Dextran/Bead Complex forms, removing cellsnot of the predetermined target population from the nutrient medium. Thepredetermined target population is collected downstream and returned tothe nutrient medium.

[0040] Clearly, many other techniques could be utilized for bothpositive and negative selection, as long as the desired affinity isprovided by the selection element.

[0041] The selection element can be simply the selection moleculeitself, or can include other components, e.g., a solid support ontowhich the selection molecule is bound. The solid support can be formedof a material that will aid in performing the selection or inmaintaining the selection molecules in a desired position or introducingand removing them from the system. For example, as described above withreference to FIG. 1, the selection molecule can be bound to iron orother magnetic particles to allow the selected cells to be easilyremoved from the system by application of a magnetic field and thencollected by removal of the magnetic field. Alternatively, the selectionmolecules can be bound onto the wall of a vessel containing the nutrientmedium, or of a chamber through which the nutrient medium flows duringthe method. Glass or other inert, impermeable beads can also be used asa solid support, as will be discussed in detail below. If beads or otherparticles are used, they can be provided in a packed configuration,through which the nutrient medium flows, or can be introduced into thesystem in a loose form, suspension, or in any desired type of array. Aswill be readily understood, a wide variety of other solid supports canbe used.

[0042] As shown in FIGS. 2-2 d, the selection element can be used in avariety of modes of operation in which nutrient media is supplied to andremoved from the system in different manners. These modes of operationrange from a selective batch culture (FIG. 2), in which nutrient mediais supplied at the beginning of cell proliferation and is neither addedto nor removed, to continuous flow or recycled flow cultures (FIGS. 2cand 2 d, respectively) in which either fresh or recycled nutrient mediaflows through the system substantially continuously. These alternativemodes will be discussed in detail below.

[0043] In a selective batch culture (FIG. 2), a nutrient medium isintroduced into a vessel, and a starting sample of cells is alsointroduced into the vessel. During cell proliferation, nutrient mediumis neither introduced nor removed. However, selected cells arephysically selected, i.e., separated from other cells in the nutrientmedium by binding to a selection element, either continuously,intermittently or following cell proliferation. These selected cells maybe cells of a target population, if positive selection is used, orunwanted cells, if negative selection is used. Dual (positive andnegative) selection can be accomplished by providing positive selectionmolecules on the surface of the vessel, beads, baffles, impellers, etc.while removing unwanted cells by negative selection. Alternately, cellsmay be positively or negatively selected outside of the culture vesseland then returned.

[0044] The selective semi-batch (2 a) and selective fed batch (2 b)modes of operation are similar to the selective batch mode with regardto positive and negative selection. The significant difference betweenthese three modes is in the treatment of the nutrient medium. While inthe batch mode the volume of the medium remains constant and the mediumis not refreshed (it may be supplemented), the semi-batch mode allowsfor a partial refreshment of spent medium with new medium and the fedbatch mode allows for an incremental increase in the medium volume overtime.

[0045] Cell growth and selection can also be performed in a continuous(FIG. 2c) or recycling (FIG. 2d) mode of operation. In these two modes,the system includes a chamber having an inlet and an outlet, andnutrient media is caused to flow through the chamber from the inlet tothe outlet. In continuous mode, new nutrient media flows through thechamber from a source or reservoir, while in recycling mode the samenutrient media is cycled through the chamber repeatedly. If desired, asystem can be configured to be run alternatively in either continuous orrecycling mode. Any desired selection element can be used in these modesof operation.

[0046] The following sections describe a bioreactor that is suitable foruse as the chamber and selection element in the “continuous” (FIG. 2c)and “recycling” (FIG. 2d) modes of operation described above. Thisdescription is intended merely as an example of one suitable type ofchamber/selection element.

[0047] Bioreactor

[0048] Referring to FIG. 3, bioreactor 10 includes two reducing unionconnectors 12, 14, mesh grids 16, 18 disposed in the orifice of eachreducing union, and a culture column 20, disposed between the two meshgrids 16, 18, containing surface activated beads 22. Mesh grids 16, 18retain the beads in the culture column 20.

[0049] Prior to use of the bioreactor, a coupling agent 11 is bound tothe activated sites 13 at the surface of the bead 22, and a selectivebinding molecule 15, selected to bind a surface antigen present onrelatively undifferentiated cells but not on relatively differentiatedcells, is in turn bound to the coupling agent 11, forming the surfacelayer shown schematically in FIG. 3a. The coupling agent is providedbetween the selective binding molecule and the activated site in orderto control the stereospecific orientation in which the selective bindingmolecule extends from the bead surface. It has been found that, byproviding the coupling agent, more desirable orientations can beobtained. However, the selective binding molecule 15 can be bounddirectly to the surface of the bead if the orientation of the selectivebinding molecule is not a concern, e.g., if enough activation sites areprovided that a sufficient number of molecules will have an orientationwhich will bind the relatively undifferentiated cells.

[0050] The manner in which the coupling agent and selective bindingmolecule are bound to the beads during manufacture of the culturecolumn, to form the structure shown in FIG. 3a, is described in detailin the “Bioreactor Setup” and “Examples” sections below.

[0051] Bioreactor Set-Up

[0052] Before cell proliferation, described in the “Bioreactor Use”section, below, can begin, it is necessary to prepare the bioreactor bybinding the coupling agent and selective binding molecule to the surfaceof the beads in the bioreactor, to form the bead surface showndiagrammatically in FIG. 3a (i.e., to prepare the beads to bind thecells to be proliferated).

[0053] The coupling agent is applied by recycling a solution of thecoupling agent through the bioreactor while the bioreactor is connectedin the configuration shown in FIG. 5 (a cycling configuration). Afterthe coupling agent has been cycled through the bioreactor for sufficienttime to cause the coupling agent to bind to substantially all of theactivated sites on the beads (see, for example, the procedure describedin Example 1, below), the column is rinsed, as shown in FIG. 6, toremove excess coupling agent which is not strongly bound to the beads.The selective binding molecule is then applied in the same manner as thecoupling agent (see FIG. 5) and the column is again rinsed (see FIG. 6).

[0054] After the coupling agent and selective binding molecule have beenapplied to the beads, a plasma or plasma solution, preferably autologousor blood type cross-matched plasma, is applied in a similar manner.Preferably, the plasma is cycled through the bioreactor for about 4-6hours. It is believed that a component of the plasma functions to coatany areas of the beads which are not coated with the other reagents,thus preventing non-specific interaction between the beads andundesirable cell populations. The effect of the plasma on non-specificinteraction is shown graphically in FIG. 8. Other reagents can be usedinstead of plasma, provided that they bind to the bead surface, do notpromote cell differentiation, and do not promote nonspecificinteraction.

[0055] Preferably, substantially all of the plasma (except for the smallportion that is apparently bound to the beads) is rinsed from the columnprior to use. However, if nonspecific interaction increases in thebioreactor during use, due to washing off of the plasma coating, it maybe desirable to introduce a small amount of plasma, or a reagent whichwould function in a similar manner, to prolong the life of the column.Also, if may be desirable to include plasma in the growth medium.

[0056] During the bioreactor set-up steps described above, the incubatoris preferably maintained at the same conditions described above for thecell proliferation process.

[0057] Bioreactor Use

[0058] To use the bioreactor described above for stem cell expansion andharvesting, the bioreactor 10 is placed in a cell proliferation system30, shown schematically in FIG. 4. Cell proliferation system 30 includesa peristaltic pump 32 to provide flow of fluid through the system, areagent reservoir 34, a sampler tube 36, a waste reservoir 38, andtubing 40 connecting these components in the illustrated arrangement.The cell proliferation system 30 is disposed within an incubator 31(e.g., an incubator commercially available under the tradename NuAireNU-2700) which is maintained at approximately 37±2 degrees C., 85-90% RHand 5% CO₂ throughout the cell proliferation process.

[0059] To use the bioreactor system, a sample containing CD34+ cells,e.g., Ficoll-Paque Gradient Purified Mononuclear Fraction (MNF) (approx.5×10⁷ mononuclear cells/ml.) from bone marrow, peripheral or cord blood,or any other source of stem cells, is fed to the bioreactor through feedline 40 a. Preferably, the peristaltic pump is operated at approximately0.89 ml./hr. during feed of the sample to the bioreactor. The reactorshould be prevented from running dry during feeding by backfilling thesample tube with a rinse solution, e.g., Iscoves Modified Dulbecco'sMedium (IMDM). The pump should be run for a period sufficient tocompletely feed the MNF and thus saturate the activated sites on thebeads with CD34 cells (or, if a sample is used which contains too fewCD34 cells to entirely saturate the activated sites, to bindsubstantially all of the CD34 cells in the sample to activated sites,after which cells resulting from cell proliferation will bind to theremaining sites). For a sample size of 1 ml. containing approximately5×10⁷ cells, this will typically take about 3 hours.

[0060] Once the sample has been fed through the bioreactor as describedin the preceding paragraph, the pump is temporarily shut off while thefeed line 40 a is connected to reagent reservoir 34 containing anutrient media solution, preferably Iscove's Modified Dulbecco's Medium(IMDM), commercially available from, e.g., GIBCO BRL Products. Othernutrient media can be used; preferably the media is a defined nutrientmedia.

[0061] The pump is then restarted, again preferably at approximately0.89 ml/hr, and cell proliferation is allowed to proceed continuouslywhile the nutrient media is fed through the bioreactor.

[0062] As soon as substantially all of the activated sites on the beadsare saturated, dividing cells will begin to flow out of the column withthe exiting media, to be harvested in the sampler tube (or any othersuitable reservoir or conduit). This continuous cell harvesting willproceed, absent mechanical failure or contamination, for the life of thecolumn, e.g., until the reagents in the bioreactor are depleted throughcolumn erosion.

[0063] The use of the bioreactor described above is in the “continuous”mode of operation shown in FIG. 2c. To use the bioreactor in the“recycling” mode shown in FIG. 2d, it would simply be necessary toprovide a conduit to route fluid from the outlet of the bioreactor backto the inlet. Because flowing the fluid through a pump may tend todeleteriously effect the cells, it may be desirable to replace the pumpsystem with a gravity feed system, or otherwise prevent cells from beingdamaged during recycling.

[0064] Process Parameters

[0065] A number of parameters can be varied to affect the rate andpurity of the cell output obtained during bioreactor use.

[0066] For example, the flow rate and dilution rate of the nutrientmedia flowing through the bioreactor during cell proliferation can bevaried over a fairly broad range. Generally, it is important that theflow rate be sufficient to provide adequate oxygen to the cells, yet notso high as to wash the reagents and/or bound cells off of the beads. Tooptimize the volume of cell output obtained, it is preferred that thedilution rate be as high as possible without causing bound cells to bewashed from the column. The relationship between dilution rate and cellconcentration is described in Principles of Fermentation Technology, P.F. Stanbury & A. Whitaker, Pergammon Press, New York, 1984, at pp.14-17.

[0067] The dimensions of the bioreactor can also be varied. Therelationship between bioreactor length and width (the aspect ratio) canbe varied to maximize control of process parameters.

[0068] The volume and purity of the initial sample fed into the columncould also be varied.

[0069] Reagents

[0070] Coupling Agents

[0071] Suitable coupling agents for binding the selective bindingmolecule to the bead surface are those agents that will bind the desiredselective binding molecule, but will not bind undesired compounds. Whenthe selective binding molecule is a biotinylated antibody, preferredcoupling agents include avidin, streptavidin, NeutrAvidin (commerciallyavailable from Pierce Chemical, Rockford, Ill.), and other avidinderivatives. NeutrAvidin is preferred because its pI (isoelectric point)is substantially neutral and thus this protein exhibits very lownon-specific binding.

[0072] Selective Binding Molecules

[0073] Preferred selective binding molecules are biotinylatedantibodies. Other suitable selective binding molecules include celladhesion molecules, a mix of lineage specific antigen receptors, or, ifno coupling agent is used, a non-biotinylated antibody (biotinylation isonly necessary in order to effect binding of the antibody to thecoupling agent).

[0074] Reagents that are suitable for biotinylation of the antibodyinclude NHS-biotin, biotin hydrazide, biotin BMCC, and other biotinderivatives. NHS-biotin is preferred, as it appears to have minimaleffect on the reactivity of the antibody. Processes for biotinylationare well known. An example of a suitable process is given below in theExamples section.

[0075] Suitable antibodies include monoclonal CD34 epitopes andpolyclonal CD34 or any uniquely identifiable cell surface antigen orbinding site for a desired cell population. Mixtures of antibodies canalso be used to enhance antibody/cell interactions, both in number andstrength of the interactions, which can allow higher flow rates to beused without cells washing off of the beads.

[0076] Other Reagents

[0077] A suitable rinse solution to rinse the culture column both afterapplication of the coupling solution and after application of thebiotinylated antibody is Dulbecco's PBS, pH 7.4. A suitable rinsesolution to rinse the column after application of the plasma is IscovesModified Dulbecco's Medium (IMDM), which is also used as the nutrientmedia to promote cell proliferation. Other suitable rinse solutions andnutrient media are known to those skilled in the art. It may bedesirable for the nutrient media to be conditioned by cell growth. Thelevel of conditioning of the media can be enhanced by recycling thenutrient media through the chamber while concurrently removing dividingcells from the chamber.

[0078] Bioreactor Materials

[0079] The reactor components (culture column, tubing, fittings, etc.)should be autoclavable, and preferably also able to withstand gammairradiation and other harsh methods of sterilization. Moreover, thereactor components should be compatible with tissue culture and shouldnot leach undesirable compounds into the culture medium. The reactorparts further should not accommodate or promote adherence of cells,e.g., by lineage specific antigen receptors, cell adhesion molecules(CAMs) on the cell surface, or secretion products of the cultured cells,unless such antigens, CAMs or secretion products are specificallyincorporated into the selection criteria for a given cell proliferationprocess.

[0080] Suitable materials that meet these criteria includepolypropylene, stainless steel, polytetrafluoroethylene (TEFLON), PFA,and other inert medical grade materials well known in the art. For thetubing, silicone may in some cases be preferred for its relatively highoxygen permeability (allowing sufficient oxygen to reach the cells atlower flow rates); in other cases polytetrafluoroethylene may bepreferred for its very low non-specific interaction potential.

[0081] The fittings which connect the bioreactor to other elements ofthe system should be able to accommodate low holdup volume, withstandminimal pressures (typically less than 10 psi), and allow for minimalconstriction of flow so as to reduce channeling and adverse fluid flowpatterns. Adverse fluid flow patterns could result in inadequate wettingof the column core, erosion of surface coatings on particles, ordisruption of cells attached to the beads.

[0082] The beads are preferably borosilicate glass beads having epoxidegroups at their surface. Such beads are commercially available from,e.g., Potters Industries, Inc., Parsippany, N.J., under the tradenameGlass Spheres A and P series.

[0083] Other bead materials, e.g., polystyrene, or surface activations,e.g., carboxyl, can be used, provided that the surface of the bead isnon-porous, to avoid trapping cells or other material in pores on thebead surface. The bead surface should also be sufficiently smooth toallow cells, compounds and particulate matter in the chamber to flowpast the surface without adhering thereto or diffusing therein. Thesurface activation can be in the form of reactive groups extending fromthe surface of the bead due to the structure of the bead material or themanner in which the surface has been chemically treated, or can be inthe form of a reactive group extending from a coating applied to thebead surface. For example, the bead could be a polypropylene or otherpolymer bead and the surface activation could be a crosslinked coating,e.g., of an amino acid. The reactive group is selected to be capable ofbinding the selected coupling agent or, if no coupling agent is used,binding the selective binding molecule itself. Preferably, the surfaceactivation includes a sufficient number and type of binding sites toallow the beads to bind 8-12 μg NeutrAvidin per gram of beads at pH 5.0(0.1 M phosphate buffer) at room temperature during a 12-16 hour cyclingprocess (e.g., the process shown in FIG. 5). The number of binding sitescan be varied, however, to suit particular column dimensions, flowrates, or other process parameters. The bond formed with the reactivegroup (by the coupling agent or by the selective binding molecule, if nocoupling agent is used) is typically covalent.

[0084] Preferably, the beads have a diameter of about 250-550 μm, morepreferably 350-450 μm. Smaller beads, when packed in the column, may notbe sufficiently far apart to allow flow of cells through the column,while larger beads may not provide sufficient available surface area toenable efficient cell interaction. The size and size distribution of thebeads can be varied, however, to vary the surface area or number ofbinding cites available for a column having given dimensions.

[0085] In some cases, it may be desirable to include a spacer zone ofnon-activated beads at the top, bottom, or a specific region of thecolumn, or mixed with the activated beads. Such a spacer zone could beused to reduce cell-to-cell interactions.

[0086] Instead of beads, any material having suitable surface activatedsites could be used, provided that the material includes sufficient openspace to allow flow of fluid therethrough at sufficient flow rates.Thus, the matrix could comprise a honeycomb, mesh, net, or othermaterial having sufficient surface area and a network of connecting openspaces through which fluid can flow. Alternatively, a fluidized ormagnetically stabilized bed could be configured to accomplish similarobjectives.

[0087]FIG. 6 shows a system 100, in a recycle mode of operation, havingboth positive and negative selection elements. To provide the positiveselection element, positive selection molecules can be attached to (a)the inner surface 102 of the reactor vessel, (b) the surfaces of theimpeller 104 in the reactor vessel, or (c) to baffles, beads or magneticparticles inserted into the liquid in the reactor (not shown). Thisattachment of the positive selection molecules to a solid support allowsthe target population captured by the molecules to be immobilized withinthe liquid system. The negative selection element is provided by a shunt106 in the recycle loop. Shunt 106 contains a selection element specificfor a predetermined population other than the target population, asdiscussed above with reference to FIG. 1a. System 100 contains growthmedium and is housed in an incubator. The system 100 is inoculated withan extract containing target cells or cells that are progenitors of thetarget population, and conditions are controlled to result in celldivision. Cells which express positive markers for the target populationare immobilized by the positive selection element, while cells whichexpress markers not expressed on the target population are captured andremoved by the negative selection element during recycling. Shunt 106can be removed and replaced as necessary.

[0088] The following section gives an example of a therapeutic use forthe target cells. The target cells can also be used in many othertherapeutic and diagnostic applications.

[0089] Therapeutic Use

[0090] As shown schematically in FIG. 7, a patient requiringimmunotherapy would first have a small volume of his or her blood drawn.This blood would then be used as described above (Bioreactor Usesection) to produce a pool of autologous HSCs, which would beadministered to the patient as an immune system booster prior to atreatment damaging the patient's immune system and/or blood formingsystem (e.g., chemotherapy), and/or as a stimulant to the patient'scompromised immune or blood forming system after the treatment.

[0091] Alternatively, a cell sample could be used to produce a pool of aselected population of differentiated cells, by charging the cells to abioreactor of the invention and supplying to the bioreactor one or moregrowth factors selected to cause the cells to differentiate to cells ofthe selected population.

EXAMPLES Example 1

[0092] CD34 HPCA-2 (Human Progenitor Cell Antigen 2) was biotinylatedwith NHS-Biotin using the following procedure:

[0093] Dialyzed 1 ml. of a 25 μg/ml stock solution of antibody against1500 ml. of a dialysis buffer, e.g., 50 mM bicarbonate buffer pH 8.5, ina Dialyzer Slide (Pierce Chemical, Rockford, Ill.) for 12-16 hours.Immediately before using, dissolved 1 mg of the NHS-biotin in 75 μlDMSO. Added 25 μl of this solution to the dialysate. Incubated at roomtemperature for 1 hour. Transferred to a Centricon-30 microconcentrator(Amicon) and spun at 14,000 cgf for 12 minutes to remove unreactedbiotin. Recovered the retentate in a 1.5 ml Eppendorf tube. Brought thevolume up to 1.5 ml with Dulbecco's PBS. Stored in the dark at about 4°C. and used within one day.

[0094] A bioreactor, as shown in FIG. 3, was then assembled as follows:

[0095] 1. Using a standard one-hole paper punch (0.25″ punch), two 0.25″diameter grids were cut from 210 μm polypropylene mesh. Carefully placedthe grids into the 0.508 cm orifices of two stainless steel reducingunions. Used 0.508 OD PFA tubing to guide the grid into place at theinner lip of the reducer.

[0096] 2. Cut a 5 cm length of 0.508 cm OD PFA tubing (PFA-T4-062-100,Cambridge Valve and Fitting) using a razor blade, taking care to make aperpendicular cut so that the tubing would lay flush against the grid inthe reducer.

[0097] 3. Assembled one of the reducing unions onto one end of thelength of tubing, using a TEFLON front and back ferrule arrangement.

[0098] 4. Loaded approximately 0.55 grams (+/−0.05 grams) of epoxyactivated borosilicate glass beads into the bioreactor, making certainthat the beads completely filled the tubing, so that no unnecessaryvoids were present.

[0099] 5. Installed the other reducing union and front and back ferruleat the other end of the tubing, seated ends and finger tightened.

[0100] 6. Installed a 2 cm length of 0.318 OD PFA tubing (PFA-T4-062-25,Cambridge Valve and Fitting) and a TEFLON front and back ferrule at each0.318 ID end of the reducing union. Finger tightened.

[0101] 7. Installed a 50 cm length of #13 Pharmed tubing (H-06485-13,Cole-Parmer) onto each 0.318 OD PFA tubing, resulting in a closed,autoclavable loop.

[0102] 8. Hand tightened all fittings. Autoclaved the bioreactor at 121°C. for 30 minutes with a 15 minute dry goods exhaust. Upon removal fromthe autoclave, transferred the bioreactor to an 80° C. drying oven forabout 3 hours to remove residual moisture.

[0103] 9. Allowed the bioreactor to cool and re-tightened all fittingswith a wrench.

[0104] 10. Placed the bioreactor on a stand and loaded the #13 Pharmedtubing into a peristaltic pump.

[0105] Next, the bioreactor was loaded with reagents (Bioreactor Set-Up)as follows:

[0106] 1. Using scissors, the #13 Pharmed Tubing was cut approximately 5cm from the lower outlet of the bioreactor. The unsheathed end of a 1″21 gauge Vacutainer Collection Needle was inserted into each of thefreshly cut ends of the #13 tubing. The sheath was then removed from theother end of the needles and used to puncture the top of a 1.5 mlEppendorf Microfuge tube containing 1.5 ml of a coupling solution (100μg NeutrAvidin in pH 5.0 phosphate buffer (0.1M) made and filtersterilized (0.2 μm) immediately prior to use). This procedure results inthe system configuration shown in FIG. 5.

[0107] 2. Started the peristaltic pump at 50% pump output (0.89 ml/hr.)so that the coupling solution was pumped up through the bioreactor, thusreducing the likelihood of air entrapment which could produce adversechanneling effects. After about half the liquid volume had been reduced,added an additional 0.5 ml of the coupling solution to ensure that thebioreactor would be adequately supplied with solution during the entirecoupling procedure. Allowed the coupling solution to recycle through thesystem loop for 16 hours.

[0108] 3. Stopped the pump and reconfigured as shown in FIG. 5a asfollows: Changed pump directional control to allow solution to be pumpeddown. Broke the recycle loop, being careful not to introduce air, byremoving one end from the 1.5 ml Eppendorf tube. Attached this end intoa presterilized feed bottle containing a rinse solution (20 ml ofDulbecco's PBS pH 7.4). Primed the feed bottle using a luer lock syringeto apply positive pressure on the sterile exhaust filter. Removed theother end from the Eppendorf tube and installed it onto a pre-sterilizedwaste bottle.

[0109] 4. Started the pump at 50% pump output (0.89 ml/hr.) and rinsedthe bioreactor with rinse solution for 3 hours.

[0110] 5. Stopped the pump. Reconfigured as shown in FIG. 5 bytransferring the feed and waste lines to the 1.5 ml of NHS-CD34biotinylated antibody solution prepared above. Started the pump at 50%pump output and ran it in this configuration for 6 hours.

[0111] 6. Stopped the pump and repeated the reconfiguration and rinsecycle described in step 3 above.

[0112] 7. Stopped the pump. Reconfigured as shown in FIG. 5, placed a1.5 ml Eppendorf tube containing 1.5 ml of autologous blood plasma intothe recycle loop. Started the pump at 50% output and recycled for 6hours.

[0113] 8. Stopped the pump.

[0114] The pump was then reconfigured for cell proliferation, i.e., tothe configuration shown in FIG. 4. Step 3 was then repeated, replacingthe rinse solution with 1 liter of Iscoves Modified Dulbecco's Medium(IMDM).

[0115] Cell proliferation then proceeded as follows:

[0116] 1 ml of fresh Ficoll-Paque Gradient Purified Mononuclear Fraction(MNF) from peripheral blood was resuspended in HBS at approximately5×10⁷ mononuclear cells/ml. Attached the MNF to the feed line butotherwise remained in the configuration shown in FIG. 4. Started thepump at 50% output. As the MNF was fed into the bioreactor, the cellswere kept in suspension to reduce the potential for clotting orclogging. To prevent the pump from running dry, backfilled the Eppendorfwith IMDM for several hours. Did not stop the pump for at least 3 hoursafter starting the MNF through the bioreactor. Stopped the pump andreinstalled the feed line to the IMDM reservoir. Placed the sampler tubeas shown in FIG. 4. Restarted the pump at 50% output. Exchanged thesampler tube daily for analysis by hemocytometer, phase and fluorescencemicroscopy.

[0117] The following results, illustrated graphically in FIG. 9, wereobtained from the cell proliferation process using the peripheral bloodsample:

[0118] MNF passed through the bioreactor=5×10⁷ mononuclear cells/ml.

[0119] MNF recovered in filtrate after 3 hours=3×10⁷ mononuclearcells/ml. (Note that mononuclear cells continued to appear in thefiltrate for 3-5 days after the initial loading. This ended after about5 days. Included in these fractions were some CD34+ cells which wereeither loosely bound or large in size, or were in the process ofdividing when introduced to the column. Only strongly bound CD34+population remained in the reactor at 50% pump output.)

[0120] During the period from 5 days after inoculation to 8 days afterinoculation, substantially no CD34 cells left the bioreactor, indicatingthat the strongly bound CD34 cells left after the initial 5 day periodwere remaining on the column and cell proliferation was not yetdetectable.

[0121] From 8 days after the initial inoculation until bioreactorfailure, roughly 100-500 CD34+ cells per day (measured by manualmicroscopy) appeared in the sampler tube. (The average number of cellscollected per day over the life of the column was 350 cells/day. Thisnumber is shown in FIG. 9 as the number collected each day, as itappeared that deviation from the average number as merely the result ofinaccuracies in the manual counting procedure used.) The harvested cellshad a birth diameter of 5+/−2 μm, much smaller than the CD34 cells whichwere immobilized on the beads in the bioreactor, the diameters of thevast majority of which ranged nominally from about 10 μm to 15 μm. Thecell type and relative frequency remained constant for 21 days beforethe bioreactor succumbed to mechanical failure.

Example 2

[0122] The procedures described in Example 1 were repeated, substitutingcord blood for the peripheral blood in the cell proliferation process.

[0123] The following results, shown in FIG. 9a, were obtained from thecell proliferation process:

[0124] MNP passed through the bioreactor=5×10⁷ mononuclear cells/ml.

[0125] MNF recovered in filtrate after 3 hours=3×10⁷ mononuclearcells/ml. (As noted above, mononuclear cells continued to appear in thefiltrate for 3-5 days after the initial loading.)

[0126] From 8 days after the initial inoculation until bioreactorfailure, as shown in FIG. 9a, roughly 500-1000 CD34+ cells/day (measuredby manual microscopy), with a birth diameter of 5+/−2 μm, appeared inthe sampler tube. The average number of cells collected per day was 750,and, for the reasons explained above in Example 1, this is the numberthat is shown for each day in FIG. 9a. Measured by flow cytometry, asample taken from the sampler tube for the period between 10 days and 13days (3 day period) contained 12,000 cells, of which 88% were CD34+cells. The cell type and relative frequency remained constant for 28days before the bioreactor succumbed to contamination.

Example 3

[0127] The effect of treating the bead surface with plasma, or,alternatively, fetal bovine serum, on non-specific interaction at thebead surface was studied. Cord blood was centrifuged under a densitygradient (Ficoll-Paque), the mononuclear fraction (MNF) was removed andconcentrated to 1×10⁶ cells in a 10 μl volume. The sample was theninjected under flow conditions of 0.89 ml/hr via a septum into abioreactor which had been prepared as described in Example 1, steps 1-7,using cross-matched human plasma in step 7. This procedure was repeatedusing a bioreactor that had been prepared as described in the samemanner, except that fetal bovine serum (FBS) was substituted forcross-matched human plasma in step 7. The procedure was then repeatedagain using a bioreactor that had been prepared as described in Example1, steps 1-6 (no plasma or FBS treatment). Cells exiting the reactor ineach case were counted in a time-wise fashion. The resultingconcentration profile was then integrated to obtain the percent of cellsrecovered. Recovery increases as nonspecific interactions decrease, andthus the results of this experiment, shown in FIG. 8, illustrate thatnon-specific interactions were greatly reduced by treating thebioreactor with plasma, but not significantly reduced by treating thebioreactor with FBS.

Example 4

[0128] Positive (+) Selective Clonogenic Expansion In A Semi-batchCulture

[0129] Positive Selection (FIG. 1)

[0130] The population selected by the selection element was thepredetermined target population. In this case the target population wascomposed of CD34 cells.

[0131] Growth Medium

[0132] Iscove's Modified Dulbecco's Medium (IMDM) (100 ml), Pen/Strep(50 μl), BSA (50 mg/ml), Insulin (50 ug/ml), Transferrin (1 mg/ml), LowDensity Lipoprotein (100 μl), 2-Mercapto-Ethanol (7 μl of 1/100solution), Flt3 (100 ng/ml), SCF (100 ng/ml), and IL-3 (20 ng/ml).

[0133] 1. Obtained a Cord Blood extract containing CD34 cells.

[0134] 2. Separated the Mononuclear Fraction (MNF) by Ficoll DensityGradient Centrifugation.

[0135] 3. Inoculated the 4 ml culture medium with 1×10⁶ MononuclearCells (MNCs)/ml.

[0136] 4. Placed the culture plate in an incubator at 37° C. and 5% CO₂.

[0137] 5. On day 7 harvested the culture and purified by positive (+)selection (see description of positive selection procedure below) forCD34 cells while saving the culture medium for step 7.

[0138] 6. Split the culture medium in half. Discarded one half andreplenished the other half with an equal volume of fresh culture medium.

[0139] 7. Returned the positive (+) selection containing CD34 cells tothe culture medium prepared in step 5 and placed the culture plate in anincubator at 37° C. and 5% CO₂.

[0140] 8. For one selection cycle using the specified medium harvestedbetween day 10 and day 15.

[0141] 9. For multiple cycles of selection repeated steps 5 through 7each 1 to 30 days.

[0142] Positive (+) Selection Procedure (Step 5, above; shown in FIG. 1)

[0143] We incubated the cells in the culture with a cocktail containinga biotintylated antibody to the predetermined target population such asbiotintylated CD34, c-kit, Lectin, etc. To this suspension Avidin wasadded and allowed to bind the biotintylated antibody. This latersuspension was thoroughly rinsed to remove any biotintylatedantibody/Avidin complex that was not interacting with the targetpopulation. To this rinsed solution a biotintylated dextran/iron complexwas added. The biotintylated dextran/iron complex reacted with theCell/Biotintylated antibody/Avidin complex. This solution was thenpassed between a strong magnet. The predetermined target population wascaptured in the magnetic field which was later removed to allow simpleharvest of the positively selected population. This procedure isdescribed in further detail below:

[0144] Materials and Supplies

[0145] Sterile Dulbecco's Physiological Buffered Saline (PBS) with 4%Fetal Calf Serum (FCS) without Magnesium or Calcium

[0146] 1.25 μg/μl Biotintylated CD34 (BD) in DPBS with 4% FCS

[0147] 1.25 μg/μl Avidin (Pierce) in DPBS with 4% FCS

[0148] Biotintylated Dextran/Iron Complex (Miltenyi) Solution

[0149] Column (Stem Cell Technologies)

[0150] Magnet (Stem Cell Technologies)

[0151] Peristaltic Pump

[0152] Biological Safety Cabinet (BSC)

[0153] 15 ml Polypropylene Tube

[0154] 37° C./5% CO₂ Incubator

[0155] 4° C. Refrigerator

[0156] Iscove's Modified Dulbecco's Medium (IMDM) with 10% FCS

[0157] Table Top Centrifuge

[0158] Culture initiated as described above.

[0159] 1. Removed a growing (as described above) culture ofhematopoietic cells from the 37° C./5% CO₂ incubator. Spun down theculture at 1000 RPM at 4° C. for 10 minutes to pellet the cells. Savedthe supernatant medium for reuse. Resuspended the pellet in 0.3 ml IMDMwith 10% FBS.

[0160] 2. To this suspension added 100 μl of the biotintylated antibodysolution and 100 μl of the Avidin solution and refrigerated at 4° C. for30 minutes with occasional gentle mixing.

[0161] 3. Added 5 ml of IMDM with 10% FBS to the suspension and mixedgently. Centrifuged at 1000 rpm for 10 minutes at 4° C. Removed anddiscarded the supernatant.

[0162] 4. Resuspended the pellet in 0.5 ml of IMDM with 10% FBS. To thissolution added 100 μl of the biotintylated dextran/iron complex andincubated at 4° C. for 30 minutes with occasional gentle mixing.

[0163] 5. Pre-rinsed the column in once in DPBS followed by 10 columnvolumes (25 ml) of DPBS with 4% FCS at a flow rate of 2 ml/minute. Afterplacing the column within the magnetic field, applied the cellsuspension to the top of the column and began pumping the solutionthrough the column at the above rate. Kept adding fresh IMDM with 10%FBS over the original solution so the column did not at any time dryout.

[0164] 6. After roughly 25 mls had passed since the cell suspension wasadded, changed the collection tube and removed the column from themagnet. Continued to add fresh IMDM as in step 5 until 25 mls has beencollected. This fraction contained the positively selected predeterminedtarget population.

[0165] 7. Spun down the collected fraction (retentate). Removed anddiscarded this supernatant. Resuspended the pellet in half the originalconditioned medium. Refreshed the remaining half volume with new growthmedium as described above and returned to the 37° C. incubator.

Example 5

[0166] Negative (−) Selective Clonogenic Expansion in a Semi-BatchCulture

[0167] Negative Selection (FIG. 1a)

[0168] The population selected by the selection element did not includethe predetermined target population. In this case the target populationwas CD34+ cells.

[0169] Growth Medium

[0170] Iscove's Modified Dulbecco's Medium (IMDM) (100 ml), Pen/Strep(50 μl), BSA (50 mg/ml), Insulin (50 μg/ml), Transferrin (1 mg/ml), LowDensity Lipoprotein (100 μl), 2-Mercapto-Ethanol (7 μl of 1/100solution), Flt3 (100 ng/ml), SCF (100 ng/ml), and IL-3 (20 ng/ml).

[0171] 1. Obtained an Umbilical Cord Blood extract containing CD34+cells.

[0172] 2. Separated the Mononuclear Fraction (MNF) by Ficoll DensityGradient Centrifugation.

[0173] 3. Inoculated the 4 ml culture medium with 1×10⁶ MononuclearCells (MNCs)/ml.

[0174] 4. Placed the culture plate in an incubator at 37° C. and 5% CO₂.

[0175] 5. On day 7, harvested the culture and purified by negative (−)selection, as described below, for CD34+ cells while saving the culturemedium for step 7.

[0176] 6. Split the culture medium in half. Discarded one half andreplenished the other half with an equal volume of fresh culture medium.

[0177] 7. Returned the negative (−) selection containing CD34+ cells tothe culture medium prepared in step 5 and placed the culture plate in anincubator at 37° C. and 5% CO₂.

[0178] 8. For one selection cycle using the specified medium, harvestedbetween day 10 and day 15.

[0179] 9. For multiple cycles of selection repeated steps 5 through 7each 1 to 30 days.

[0180] Negative (−) Selection Procedure (Step 5 above; shown in FIG. 1a)

[0181] We incubated the cells in the culture with a cocktail containingselection molecules (antibodies to T-cell surface antigens) linked to ananti-dextran molecule. The cell suspension was then passed through acolumn containing glass beads coated with dextran. Cells with noantibody/anti-dextran were allowed to pass through the column and werecollected as the predetermined target population while those whichcomplexed were captured.

[0182] Materials and Supplies

[0183] ⅜ inch Teflon tubing approximately 2″ in length

[0184] ⅛ inch Teflon tubing approximately 2″ in length

[0185] {fraction (1/16)}0 inch ID flexible TYGON tubing approximately 1″in length

[0186] ⅜ to ⅛ inch Stainless Steel Reducer with Teflon Furrel Inserts

[0187] ⅜ inch diameter Polypropylene Mesh with gridded 210 um openings

[0188] 1 gram of Epoxy Glass Beads 425 um nominal diameter

[0189] dH₂O

[0190] 1 gram of Dextran (Sigma, Tissue Grade)

[0191] 10 ml 0.1M pH 5.0 Sodium Phosphate Buffer

[0192] Sterile Dulbecco's Physiological Buffered Saline (DPBS) with 4%Fetal Calf Serum (FCS) without Magnesium or Calcium

[0193] Antibody/anti-dextran cocktail composed of the followingselection molecules:

[0194] CD2, CD3, CD14, CD16, CD19, CD24, CD56, CD66b and glycophorin Aat concentrations between 0.5 and 1.25 μg/μl each.

[0195] Ring Stand

[0196] Tubing Clamp

[0197] Autoclave

[0198] Biological Safety Cabinet (BSC)

[0199] 15 ml Polypropylene Tube

[0200] Sterile Filter and Syringe

[0201] 37° C./5% CO₂ Incubator

[0202] 4° C. Refrigerator

[0203] Iscove's Modified Dulbecco's Medium (IMDM) with 10% FCS

[0204] Table Top Centrifuge

[0205] Culture initiated as described above.

[0206] 1. Assembled the ⅜″ and ⅛″ Teflon tubes with one reducing nutcomplete with Teflon furrels. Prior to inserting the ⅜″ Teflon tube,placed the polypropylene grid between the tubing and the inner flange ofthe fitting. Hand tightened the fitting, installed the {fraction(1/16)}″ ID Tygon tubing on the ⅛″ OD Teflon, and wraped loosely withfoil to autoclave with preparation in step 2. (Note: one end of thetubing has no reducing nut.)

[0207] 2. Weighed out 1 gram of epoxy glass beads into a 15 mlpolypropylene tube. Submersed the beads in about 2 ml of dH₂O. Looselyinstalled the cap and placed in the autoclave with contents of step 1.

[0208] 3. After autoclaving, transferred the sterile materials fromsteps 1 and 2 to the Biological Safety Cabinet.

[0209] 4. Prepared a sterile dextran solution by adding 2 grams ofdextran to 10 mls of the pH 5 Phosphate Buffer (0.1M) and sterilefiltering through a syringe.

[0210] 5. Removed the dH₂O from the beads and replaced the liquidcontents with approximately 2 ml of the sterile dextran solution.Allowed this solution to incubate at 37° C. for at least four hours.

[0211] 6. Removed the sterile dextran solution and with repeat rinsesreplaced the dextran solution with sterile DPBS containing 4% FCS.Allowed this solution to incubate for about 90 minutes at roomtemperature.

[0212] 7. Unwrapped the column assembly and installed a tubing clamponto the {fraction (1/16)}″ Tygon tubing. Tightened the clamp tocompletely restrict the pathway. Perpendicular to the working surface,clamped the column so that the end with the reducing nut, ⅛″ Teflon,{fraction (1/16)}″ Tygon and tube clamp aimed downward.

[0213] 8. Using a pipette, transferred the DPBS solution containing theDextran coated Glass Beads into the top, open end, of the columnassembly. Adjusted the tube clamp so that the liquid flowed down throughthe column.

[0214] 9. Washed the column with approximately 3 ml of IMDM with 10%FBS. The column was now ready to receive cells.

[0215] 10. Removed a growing (as described above) culture ofHematopoietic cells from the 37° C./5% CO₂ incubator. Spun down theculture at 1000 RPM at 4° C. for 10 minutes to pellet the cells. Savedthe supernatant medium for reuse. Resuspended the pellet in 1 ml IMDMwith 10% FBS.

[0216] 11. To this suspension added 200 μl of the antibody/anti-Dextrancocktail and refrigerated at 4° C. for 30 minutes with occasional gentlemixing.

[0217] 12. Applied the suspension to the column prepared in steps 1-9,collecting the permeate below in a 15 ml polypropylene centrifuge tube.

[0218] 13. Rinsed the suspension with at least 3 ml of IMDM with 10% FBSwhile continuing to collect the permeate.

[0219] 14. Centrifuged the permeate. Removed and discarded thissupernatant. Resuspended the pellet in half the original conditionedmedium. Refreshed the remaining half volume with new growth medium asdescribed above and returned to the 37° C. incubator.

[0220] Results of Examples 4 and 5

[0221] The results of the experiments performed in Examples 4 and 5 areshown graphically in FIGS. 11 and 11a. FIG. 11 represents the relativeincrease in CD34+ cells in duplicate samples of a representative cordblood extract. All data points are from the same extract. All cultureswere manipulated on day 7 in this experiment. The unselected sample wassplit and replenished with ½ fresh medium to mimic medium conditions inthe other samples. Inefficiencies in the separation procedures have beenaccounted for. FIG. 11a shows the relative increase in leukocytes underthe same conditions in the same experiment.

[0222] Other Embodiments

[0223] Other embodiments are within the following claims.

[0224] For example, while the method of the invention has been describedin connection with the expansion of a population of relativelyundifferentiated cells, preferably HSCs, the method and system can beused to expand other cell populations. As shown in FIG. 10, relativelydifferentiated cells could be expanded, e.g., in a bioreactor downstreamfrom a bioreactor used to expand HSCs. While most or all differentiatedcells are not renewable, the cells could be expanded for a limitednumber of generations, or, if the cells are renewable, conceivably foras many expansions as are possible for HSCs.

1. A method of selective expansion of a predetermined target populationof cells, said method comprising: introducing a starting sample of cellsinto a growth medium; causing cells of said predetermined target cellpopulation to divide; and contacting the cells in the growth medium witha selection element, comprising a plurality of selective bindingmolecules with specific affinity either for target cells or for a firstpopulation of non-target cells, so as to select cells of saidpredetermined target population from other cells in the growth medium.2. The method of claim 1 wherein said selective binding molecules arespecific for said target cells.
 3. The method of claim 1 wherein saidselective binding molecules are specific for non-target cells.
 4. Themethod of claim 2, further comprising contacting the starting cells witha reverse selection element comprising selective binding molecules withspecific affinity for a second population of non-target cells.
 5. Themethod of claim 1, further comprising removing cells from said growthmedium.
 6. The method of claim 5, wherein the removed cells are cells ofsaid predetermined target cell population.
 7. The method of claim 5wherein the removed cells are non-target cells.
 8. The method of claim 1wherein said starting sample of cells includes said target cells, andsaid expansion is clonogenic.
 9. The method of claim 1 wherein saidstarting sample of cells includes progenitors of said target cells. 10.The method of claim 1, further comprising removing the growth mediumprior to said contacting step.
 11. The method of claim 1, wherein saidselection element comprises a solid support to which said selectivebinding molecules are bound.
 12. The method of claim 11 wherein saidgrowth medium is disposed in or caused to flow through a chamber. 13.The method of claim 12 further comprising causing said growth medium torecycle through said chamber, flowing from an inlet, through saidchamber, to an outlet of said chamber, and returning from the outlet tothe inlet via a conduit.
 14. The method of claim 2 wherein saidselective binding molecules bind to a cell surface antigen on cells ofsaid predetermined target cell population but not on cells not in saidtarget cell population.
 15. The method of claim 14 wherein saidselective binding molecule is a biotinylated antibody specific for anantigen on the surfaces of said cells of said predetermined target cellpopulation.
 16. The method of claim 3 wherein said selective bindingmolecules bind to a cell surface antigen that is on cells of thenon-target cell population but not on cells of said target cellpopulation.
 17. The method of claim 11 further comprising the step ofcausing plasma to bind to regions of said solid support on whichselective binding molecules are not present.
 18. The method of claim 17wherein said plasma is autologous plasma.
 19. The method of claim 17wherein said plasma is a type-matched allogeneic plasma.
 20. The methodof claim 11, further comprising the step of contacting the solid supportwith an agent that is capable of binding to regions of said solidsupport on which said selective binding molecules are not present toprevent non-specific interaction between said regions and materialscontacting said solid support.
 21. The method of claim 1 wherein saidgrowth medium is conditioned by cells present in the growth medium. 22.The method of claim 28 further comprising causing the growth medium torecycle through a fluid flow loop, thereby enhancing the level ofconditioning.
 23. The method of claim 29 further comprising concurrentlyremoving dividing cells from the fluid flow loop during recycling. 24.The method of claim 1, further comprising regulating the oxygensaturation of the growth medium to be from 0% to 20% relative to thesolubility of oxygen in said fluid at equilibrium with air at 37° C. and1 atm pressure.
 25. A system for continuous selective expansion of apredetermined target population of cells, comprising: a growth mediumfor supporting cell division; a chamber for receiving said growthmedium; and a selection element, comprising a plurality of binding sitesbearing a selective binding molecule having a specific affinity forcells of said predetermined target cell population, positioned tocontact said growth medium during or after cell division.
 26. A systemfor continuous selective expansion of a predetermined target populationof cells, comprising: a growth medium for causing cell division; achamber for receiving said growth medium; and a selection element,comprising a plurality of binding sites bearing a selective bindingmolecule having a specific affinity for cells of cell populations otherthan said predetermined target cell population and having substantiallyless affinity for cells of said predetermined target cell population,said selection element being positioned to contact said growth mediumduring or after cell division.
 27. A method of selective expansion of apredetermined target population of cells, said method comprising:introducing fluid containing a plurality of cells into a growth medium;causing cells of said predetermined target cell population to divide;and selecting cells of said predetermined target population from othercells in the growth medium; wherein said causing and selecting steps arecarried out substantially simultaneously.
 28. A method of enhancing theimmune function of a patient, said method comprising introducing asample containing relatively undifferentiated cells into a growthmedium; causing said relatively undifferentiated cells to divide;selecting relatively undifferentiated cells from other cell populationspresent in said growth medium; and administering a portion of theselected relatively undifferentiated cells to the patient.
 29. Themethod of claim 28 wherein the sample is autologous.
 30. The method ofclaim 29 wherein the sample comprises blood or bone marrow of saidpatient.
 31. The method of claim 28 wherein said sample is allogeneic.32. A method of treating a patient in need of cell infusion, comprising:introducing a sample containing relatively undifferentiated cells into agrowth medium; introducing into said growth medium one or more growthfactors selected to cause said relatively undifferentiated cells todifferentiate to form a predetermined population of cells; causing cellsin said growth medium to divide; selecting cells of said predeterminedpopulation from other cells present in said growth medium; andadministering cells of said predetermined population to the patient. 33.The method of claim 32 wherein said second population of cells compriseslymphoid precursors, myeloid precursors, or erythroid precursors.
 34. Amethod of reducing negative feedback during selective clonogenicexpansion of relatively undifferentiated cells comprising immobilizing apopulation of relatively undifferentiated cells on a solid support,providing a nutrient-containing medium to said cells, continuouslyremoving waste products of said cells from contact with saidimmobilized, relatively undifferentiated cells, while continuouslyremoving relatively undifferentiated cells immediately after they areformed.
 35. A method of treating a patient in need of a therapeuticcompound, said method comprising: (a) providing a sample containing afirst population of cells which produce said therapeutic compound; (b)passing a portion of the sample through a chamber containing a solidsupport capable of supporting a plurality of living cells adheredthereto, wherein said support comprises a plurality of binding siteswith specific affinity for said first population of cells so that aportion of said cells bind to said binding sites; (c) causing mediumcontaining nutrients to flow through said chamber to cause said boundcells to produce said compound; (d) harvesting said compound from thechamber; and (e) administering said compound to the patient.
 36. Amethod of making a bioreactor for continuous selective clonogenicexpansion of a predetermined population of cells, said methodcomprising: (a) providing a chamber having an inlet, an outlet, and apassage for fluid flow from the inlet to the outlet, and, disposed inthe chamber, a solid support having a plurality of activated sites; (b)binding a selective binding molecule with specific affinity for saidpredetermined population of cells to the activated sites to enable theactivated sites to support a plurality of living cells adhered thereto;(c) introducing into the chamber an agent which is capable of binding toregions of said solid support on which said selective binding moleculesare not present and preventing non-specific interaction between saidregions and materials subsequently introduced into the chamber; (d)causing fluid containing a plurality of cells of said cell population toflow through said chamber, so that a portion of said cells bind to saidbinding sites; and (e) introducing a growth medium to the chamber. 37.The method of claim 36 wherein said agent is plasma.
 38. The method ofclaim 37 wherein said plasma is autologous with respect to the cellpopulation.
 39. The method of claim 37 wherein said plasma is atype-matched allogenic plasma.